Staphylococcus epidermidis and Staphylococcus aureus are the leading causes of indwelling-device related infections, affecting up to 400,000 patients/year in the United States. In the body, these pathogens form biofilms on medical implants that are intrinsically resistant to antibiotics and attack by the immune system. Our overall goal is to develop drugs that specifically target the biofilm mode of growth. This represents an innovation over existing antibiotic therapies that target planktonic bacteria and are not effective in eradicating biofilm bacteria. Our strategy is to screen for small molecule compounds that reduce the intrinsic resistance of Staphylococcal biofilms to antibiotics. These compounds will sensitize biofilm bacteria to antibiotics that are in clinical use. In Phase I, we will identify and validate gene targets for small molecule drug discovery. The specific aims are as follows: Aim 1. Develop a high-throughput screen for identifying mutants defective in intrinsic antibiotic resistance in biofilms. Aim 2. Identify S. epidermidis mutants defective in intrinsic antibiotic resistance in biofilms. Aim 3. Evaluate mutants in a panel of in vitro assays, identify transposon insertion sites, and prioritize mutants for target validation studies. Aim 4. Validate genes identified in Aim 2 and Aim 3 as potential targets for small molecule drug discovery. In Phase II, we will develop screens for validated targets and use them to identify compounds that sensitize Staphylococcal biofilms to antibiotics. We will develop these compounds into innovative drugs that will be used in combination with clinically relevant antibiotics to eradicate biofilm infections.